SUPPLEMENTARY INFORMATION

Transcription

1 Materials and Methods Transgenic Plant Materials and DNA Constructs. VEX1::H2B-GFP, ACA3::H2B-GFP, KRP6::H2B-GFP, KRP6::mock21ts-GFP, KRP6::TE21ts- GFP and KRP6::miR161ts-GFP constructs were generated through cloning of an overlapping PCR fragment containing the promoter sequence and the histone H2B sequence without stop codon with the primers specified in Supplementary Table 3. The overlapping PCR fragments generated were cloned in a pentr-d-topo gateway entry vector (Invitrogen). All the confirmed positive entry vectors were recombined into a promoterless version of the gateway destination vector pb7fgwg2,0 generated through removal of the 35S promoter region with the restriction enzymes SpeI and SacI and self-ligation of the resulting linearized vector. MGH3::MGH3-GFP, MGH3::MGH3-GFP-VEX1 3 UTR and MGH3::MGH3-GFP-TE 3 UTR were generated through sequential In-Fusion cloning of the MGH3 promoter plus the MGH3 coding sequence fragment without stop codon with the primers specified in Supplementary Table 3. The PCR fragment generated was cloned into the KpnI unique restriction site generated in a pmdc107 gateway destination vector modified through digestion with the restriction enzyme KpnI in order to remove the ccdb region. The digested vector was self-ligated in order to create a unique KpnI restriction site. This digestion leaves a unique SacI restriction site on the 3 UTR of the vector between the stop codon of mgfp6 and the nos terminator that was used to In-Fusion clone a 315 bp fragment of VEX1 (AT5G62850) sense coding region or a 315 bp fragment of Athila2 sense coding region with the primers specified in Supplementary Table 3. KRP6::mock22ts-trGFP, KRP6::TE21ts-trGFP, KRP6::miR173-trGFP, VCK1::mock22ts-trGFP, VCK1::TE21ts-trGFP and VCK1::miR173-trGFP were generated by cloning a trgfp fragment into pentr-d-topo (Invitrogen) with the primers specified in Supplementary Table 3. This allowed the cloning of a 420bp fragment from mgfp6 and the generation of a unique KpnI restriction site before the trgfp fragment. This KpnI unique restriction site was used to linearize a positive trgfp pentr-d-topo clone and in-fusion clone the KRP6 and VCK1 promoters with the different srna target sites amplified with the PCR primers specified in Supplementary Table NATURE PLANTS 1

2 3. Positive clones containing the KRP6 and VCK1 promoter with the srna target sites and the trgfp fragment were LR recombined into a pbgw,0 gateway destination vector. The KRP6::2b-RFP clone was generated by cloning of the viral silencing suppressor from a provided 2b plasmid (Feng Qu, The Ohio State University) into pentr-d-topo (Invitrogen) with the primers specified in Supplementary Table 3, which allowed both the creation of an unique KpnI restriction site right after the cloning site of the pentr-d-topo vector and the cloning of 2b without a stop codon. A confirmed positive clone was digested with KpnI, linearized and used for In-Fusion cloning of the KRP6 promoter with the primers specified in Supplementary Table 3. A confirmed positive entry clone was recombined into a promoterless version of the gateway destination vector pb7rwg2,0 generated through digestion of the 35S promoter region with the restriction enzymes SpeI and SacI and self-ligation of the resulting linearized vector. Analysis of microarray expression levels Developmental analysis of the level of steady state transcript accumulation in Supplemental Figure 1B was extracted from the Pollen Transcriptome Navigator ( which contains ATH1 microarray data for unicellular, bicellular, and tricellular pollen transcriptomes extracted from 1. Sperm and whole pollen transcriptome data for Supplemental Figure 3D is extracted from 2. 2 NATURE PLANTS

3 SUPPLEMENTARY INFORMATION Supplementary Figures Supplementary Figure 1. Identification of pollen vegetative cell-specific promoters (a) Experimental scheme to identify promoters that are specific to the vegetative cell and only expressed after pollen mitosis I. (b) Microarray steady-state mrna expression levels though pollen development. (c) The ACA1 and VEX1 promoters are specific to the vegetative nucleus and express late in pollen development. Each promoter drives the expression of an H2B-GFP fusion protein. DAPI marks the nuclei while RFP shows the background fluorescence levels. NATURE PLANTS 3

4 Supplementary Figure 2. Isolation of pure unicellular microspores The KRP6::H2B-GFP homozygous line was used for gradient centrifugation and microspore isolation. These microspores do not fluoresce GFP and only have one point of DAPI (one nucleus), in contrast to the 2-3 visible nuclei and GFP fluorescent vegetative nuclei detected in mature pollen. 4 NATURE PLANTS

5 SUPPLEMENTARY INFORMATION Supplementary Figure 3. Vegetative cell-specific expression and accumulation of RNAi pathway mrnas and proteins (a) Epifluorescence analysis of an AGO1 protein fusion to GFP under control of the endogenous AGO1 promoter. This transgene is from 3. The pollen grain also has the vegetative cell nucleus specific Act11pro::H2B-mRFP transgene from 4. (b) Confocal slice of an AGO1pro::AGO1-GFP pollen grain (top) and a sibling pollen grain that did not inherit the segregating transgene (bottom). The AGO1 protein accumulates in the pollen vegetative cell cytoplasm and nucleus but not in the sperm cells, which appear as two distinct dark shadows within the cytoplasm of the vegetative cell. (c) Analysis of a pollen grain with a transgene that has the DCL4 promoter driving H2B-GFP (DCL4pro::H2B-GFP). The pollen grain also has the vegetative cell nucleus specific Act11pro::H2B-mRFP transgene as in panel a. The DCL4 promoter is expressed from the pollen vegetative nucleus. (d) Ratio of sperm cell steady state mrna accumulation to whole pollen accumulation. Transcripts below 1.0 are vegetative cell enriched. NATURE PLANTS 5

6 Supplementary Figure 4. Comparison of pollen small RNA-sequencing libraries (a) Total microrna accumulation (mirbase release 21) normalized in reads per million. The libraries are not direct biological replicates, as the method of pollen harvesting and the genotypes differ between the wt Col pollen sequenced in 5 and this study. 6 NATURE PLANTS

7 SUPPLEMENTARY INFORMATION (b) Endogenous TE 21-22nt sirna production. TE 21-22nt sirna reads per million are reduced by roughly half in the dcl2/dcl4 double mutant. (c) Accumulation of microrna161 (black, above the X-axis) and the mock 21nt target site targeting small RNAs (red, below the X-axis). (d) Accumulation of microrna173 (black, above the X-axis) and the mock 22nt target site targeting small RNAs (red, below the X-axis). The accumulation of mirna173 is lower in genotypes with the mir173 target site transgene, suggesting that AGO cleavage of the mir/target mrna complex reduces a limited pool of microrna173. (e) Accumulation of sirnas that target the 3 UTRs of the GFP transgenes from Figure 4D-E. The Athila TE 3 UTR is shown in black above the X-axis and the Vex1 3 UTR is shown in red below the X-axis. For parts b-e, normalization was performed using reads per million (RPM)(left Y-axis) and reads per million micrornas (RPMM)(right Y-axis). Supplementary References 1 Honys, D. & Twell, D. Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biol 5, R85 (2004). 2 Borges, F. et al. Comparative transcriptomics of Arabidopsis sperm cells. Plant Physiol 148, (2008). 3 McCue, A. D., Nuthikattu, S., Reeder, S. H. & Slotkin, R. K. Gene expression and stress response mediated by the epigenetic regulation of a transposable element small RNA. PLoS Genet 8, e (2012). 4 Rotman, N. et al. A novel class of MYB factors controls sperm-cell formation in plants. Curr Biol 15, (2005). 5 Slotkin, R. K. et al. Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell 136, (2009). NATURE PLANTS 7